A review study on the effect of Iranian herbal medicines against in vitro replication of herpes simplex virus.

OBJECTIVE
There are a number of published data indicating in vitro anti-HSV activity of some of Iranian herbal extracts with no systematic review to discuss these results. Therefore, this article was aimed to review and discuss the methods carried out and the phytochemistry and bioactivity of the extracts used and also conclusions provided in these publications.


MATERIALS AND METHODS
Published articles both in English (from Medline, Science Direct, EMBASE, Scopus, Pro Quest, Google scholar, Cochrane Library) and in Persian (from SID, Iran Medex and Magiran) databases, from 1966 to October 2014 were incorporated in this review. The in vitro studies that lacked CC50, IC50, were excluded.


RESULTS
Only 42 published reports were found to examine Iranian herbs against HSV replication in vitro. Seventeen out of 42 studies in which 23 kinds of medicinal plants were subjected to crude extraction were included. The review of data showed that some of the herbal extracts including Hyssopus officinalis methanolic extract, Melissa officinalis aqueous extract, Quercus persica L. hydroalcoholic extract and Securigeras ecuridaca methanolic extract with selective index (SI) of 234, 877, >778 and 250, respectively were highly effective against HSV in vitro.


CONCLUSION
More comprehensive studies using more advanced methods are needed to be done to achieve promising anti-HSV agents from the bioactive compounds isolated from these herbs.


Introduction
Belonging to the family of Herpesviridae and subfamily of Alphaherpesvirinae, herpes simplex virus type-1 (HSV-1) and type 2 (HSV-2) are two of the most common human pathogens. Infections caused by these viruses are public health concern worldwide. These infections range from inapparent to severe life threatening infections such as encephalitis (Koelle and Corey, 2008). Although HSV-1 and HSV-2 are usually transmitted via different routes and infections caused by these two viruses involve different areas of the body, there is a great deal of overlapping features between the epidemiology and clinical manifestations of their infections. It is generally accepted that HSV is involved in oral diseases as HSV-1 is associated with recurrent aphthous ulceration (Malvy et al., 2007). HSV-1 infection commonly occurs in the mouth and lips resulting in gingivostomatitis (Malvy et al., 2007).
During the past two decades, the mechanisms of replication and pathogenesis of HSV-1 and thus the potential antiviral targets in this virus have been widely understood leading to development of antiviral compounds that target these mechanisms (Koelle and Corey, 2008). The antiviral drugs that are most commonly used to treat HSV-1 infections are nucleoside analogues such as Acyclovir (ACV) and Penciclovir of which Acyclovir is widely used as a drug of choice (Villarreal, 2003). However, a major problem associated with the use of ACV, is the development of drug-resistant HSV strains, particularly in AIDS patients (Elion, 1993). This kind of drug-resistance may occur after long-term treatment and is mainly due to mutations in HSV-1 thymidine kinase and/or DNA polymerase genes (Hill et al., 2009).
As potential alternatives to these agents, antiviral agents from medicinal plants with effective compounds exhibiting different modes of action against HSV infections are urgently needed. These natural active compounds are potential source of novel antiviral agents that could be widely used against these viruses. Some of these compounds have been identified to be active against HSV infections. They have high chemical diversity with biochemical specificity and have biologically active small molecules with drug-like properties that can be absorbed and metabolized in the body (Kitazato et al., 2007). Furthermore, they are easily available and no laborious pharmaceutical work is needed for their synthesis. Therefore, these natural products may be good candidates for development of special anti-HSV agents (Girish and Pradhan, 2008;Li et al., 2009).
To the best of our knowledge, so far, no systematic review has been published regarding Iranian medicinal plants. Therefore, this article was aimed to review and summarize published results reporting Iranian medicinal plants with in vitro activity against HSV replication. This is to provide some recommendations for future research.

Materials and Methods
To provide this systematic review, a number of databases including Medline, Science Direct, EMBASE, Scopus, Pro Quest, Google scholar, Cochrane Library Database and Persian databases such as SID, Iran Medex and Magiran, from 1966 to October 2014 were searched. The terms Herpes simplex virus, anti-viral, plant (herb), Iran, and natural or herbal medicine, were used as keywords. The databases used were limited to those Iranian articles published both in English and in Persian. In this review, in-vitro studies lacking CC 50 , IC 50 , were excluded. All selected articles were studied by two reviewers to examine inclusion criteria and including common and scientific names of herbs, and their outcome.

Results
Using on line database search, only 42 published reports that examined Iranian herbs for in vitro inhibition of HSV replication were found. In this systematic review, 17 studies in which 23 kinds of medicinal plants were subjected to crude extraction were included and the remaining that used no standard methods to evaluate CC 50 , IC 50 , and SI, were excluded. The review of data showed that some of the herbal extracts including Hyssopus officinalis methanolic extract, Melissa officinalis aqueous extract, Quercus persica L. hydro alcoholic extract and Securigeras ecuridaca methanolic extract with SI of 234, 877, >778 and 250, respectively were highly effective against HSV, in vitro (Astani et al., 2014;Behbahani, 2009;Behbahani et al., 2013a;Karimi et al., 2013). Data of these published studies are summarized in Table  1. Since each extract with SI≥4 should be considered as a potential antiviral agent (Tsuchiya et al., 1985), the characteristics of some of the extracts isolated from some Iranian medicinal plants are discussed below.

Avicenna marina
Avicenna marina (commonly known as grey mangrove or white mangrove), is one of the mangrove species plants which is native to Southern Africa (Duke, 1991). This plant grows in Hara forests of Persian Gulf and is used in Persian folk medicine. Leaf extracts of A. marina contain compounds such as iridoid glucosides, fatty acids, sterols and hydrocarbons (Hogg and Gillan, 1984;König and Rimpler, 1985).
Based on the results of a study, the methanol extract of this herbal medicine showed higher antiviral activity against HSV, in vitro. The most polar fraction (D) of the methanol extract showed significant anti-HSV activity. This fraction inhibited the HSV replication following entry of the virus into the target cells. The phytochemical examinations of this extract have shown that fraction D contains flavonoid compounds. It has been shown that A. marina contains active flavonoid compounds which inhibit the replication of HSV (KOS strain) following penetrating into the target cells (Namazi et al., 2013).
Behbahani et al showed that crude methanol extract of A. marina and the corresponding fractions LMEG and luteolin inhibit HSV-2 replication with selective index (SI) of 25, 25 and 5.3, respectively. The results indicated that LMEG was the most active sub-fraction of A. marina with inhibitory effect on the early stage of HSV-2 replication (Behbahani et al., 2013b). Zandi et al showed that crude hot glycerin extract of fresh leaves of A. marina has antiviral activity against HSV-1 replication, in vitro, mostly before adsorption of the virus to the cells (Zandi et al., 2009).

Aloe vera
Aloe vera is a member of the Liliaceae family. Historically, it has been used for medical purposes such as treatment of wounds and burns (Perfect et al., 2005). This medicinal plant is the source of two herbal preparations namely, aloe gel and aloe latex. Aloe gel is often referred to a clear gel or mucilaginous substance produced by parenchymal cells located in the central region of its leaves. Diluted aloe gel is commonly referred to as A. vera extract. The main components of this gel are water (99%), monosaccharides, and polysaccharides (25% of the dry weight of the gel) (Shelton, 1991). There are some published reports indicating that the extracts of A. vera and particularly its anthraquinone compounds have antiviral activity (Andersen et al., 1991;Sydiskis et al., 1991). It has been shown that crude hot glycerine extract of Aloe vera gel, grown in southwest of Iran, Bushehr, inhibits HSV-2 replication, both before and after attachment to the Vero cell line (Zandi et al., 2007).

Hyssopus officinalis
Hyssopus officinalis is a member of the Lamiaceae family. Some members of this family such as Origanum vulgare are known to be poisonous and traditionally used as an arrow poison. Growing in different geographical regions of Iran, H. officinalis, mainly its leaves, has been used in the traditional Iranian medicine (TIM) for treatment of various ailments (Kazazi et al., 2007). Methanolic extract of the leaves of H. officinalis, has exhibited remarkable activity against both wild type and resistant strains of HSV, in vitro. The extract, also, significantly inhibited plaque formation of both of the two strains of HSV-1 in Vero E6 cells with minimal cell cytotoxicity (CC 50 = 960 μg/ml). An oral dose of the extract (125 mg/kg) significantly delayed the onset of HSV-1 infection by over 50%. It also increased the mean survival time of infected mice by 55-65% compared to the non-treated mice (p<0.05). The mortality rate for mice treated with the extract was also significantly reduced by 90% in comparison with the non-treated mice that exhibited 100% mortality (Behbahani, 2009).

Melissa officinalis
Leaves of Melissa officinalis (lemon balm), a member of the Lamiaceae family (Saller et al., 2001;Wolbling and Leonhardt, 1994) contain polyphenols such as rosmarinic acid and flavonoids (Carnat et al., 1998). M. officinalis has been evaluated for its activity against HSV, in vitro (Geuenich et al., 2008;Nolkemper et al., 2006). Aqueous extract of M. officinalis has been shown to interact directly with free viral particles of two acyclovir-resistant and one acyclovirsensitive strains of HSV-1 at low IC 50 values of 0.13, 0.23 and 0.4 μg/ml and high selectivity indices of 2692, 1522 and 875, respectively with inhibiting attachment of these strains to the host cells in a dose-dependent manner. The results of this work indicated that anti-HSV-1 activity of Melissa extract was mainly due to its rosmarinic acid contents (Astani et al., 2014).

Zataria multiflora
Zataria multiflora Boiss (Avishan-e-Shirazi in Persian and Sa'atar or Zaatar in ancient Iranian medical books) is a thymelike plant and a member of Lamiaceae family that wildly grows in central and southern parts of Iran (Amin, 1991). It is used in traditional folk remedies for its antiseptic, analgesic (painrelieving) and carminative (anti-flatulence and intestine soothing) properties (Amin, 1991;Mozaffarian, 1996). In addition to antibacterial activity (Eftekhar et al., 2011), essential oil of Zataria multiflora Boiss has been shown to have significant inhibitory effect on HSV-1 replication with IC 50 values and selectivity indices of 0.0059% and 11.7, respectively (Mardani et al., 2012).

Quercus persica
Q. persica "oak" is a tree of which about 600 species exist around the world. Quercus species contain high levels of tannins in both hydrolysable and condensed (Rakić et al., 2007) forms. Oak fruit (acorn) comprises starch, protein, oil, and tannins with high amounts of amylose, amylopectin, high molecular weight and viscosed substances (Saffarzadeh et al., 1999). This fruit is useful in treatment of anemia, diarrhea and is used in livestock feeding. There are some published reports indicating antiviral (Karimi et al., 2013;Muliawan et al., 2006), antibacterial (Khosravi and Behzadi, 2006), and antiinflammatory (Kaur et al., 2004) potentials of Quercus species. These effects have been attributed to its phenolic contents, particularly to its flavonoid and tannin compounds (Chang et al., 2002). It has been suggested that tannin-based components of oat fruits have remarkable effect on virus replication with different mechanisms and with the minimum cytotoxicity (Buzzini et al., 2008). In our previous study, Q. persica L. had highly anti-HSV-1 activity and very low cytotoxicity in vitro, as exhibited no cytotoxic effect on this cell line up to concentration of 200 mg/ml. Using inhibition assay, this extract inhibited HSV-1 replication by reduction of its cytopathic effect (CPE) before and after cellular attachment in concentrations of 1.02 and 0.257mg/ml, respectively (Karimi et al., 2013).

Thymus kotschyanus
Lamiaceae (formerly Labiatae) is one of the most important plant families in which Thymus with about 215 species, is a significant genus (Zaidi and Crow, 2005). The plants of this genus are well known as medicinal plants because of their biological and pharmacological properties. Fourteen species of this genus are represented in Iranian flora, of which T. daenensis Celak, T. kotschyanus Boiss, and T. Hohen are widely used as medicinal plants. Infusion and decoction of aerial parts of these species are used in Iranian traditional medicine (ITM) as tonic, carminative, digestive, antispasmodic, anti-inflammatory, anti-tussive and expectorant and for the treatment of common colds (Zargari, 1990). Aqueous extract of T. kotschyanus has been shown to inhibit HSV-1 in vitro with IC 50 value and selectivity indices of 100μg/ml and 8, respectively (Farahani, 2013).

Curcuma longa
Curcuma longa L. has been used in Asian traditional medicine (ATM) for a long time to treat gastrointestinal upset, arthritic pain, urinary tract infection, and liver ailments (Dixit et al., 1988;Luper, 1999). One of its main component, Thcurcumin has been shown to have several pharmacological activities such as anti-inflammatory, anti-oxidant, antimicrobial properties (Maheshwari et al., 2006), and antiviral activity against hepatitis B virus (HBV) by decreasing the transcription of HBV X (HBx) gene (Kim et al., 2009). Antiviral activity of different concentrations of curcumin, galliumcurcumin and Cu-curcumin were tested against HSV-1 and the results showed that curcumin and its new derivatives had remarkable antiviral effects on replication of this virus in cell culture. The IC 50 values of curcumin, gallium-curcumin and Cucurcumin were 33.0, 13.9 and 23.1 µg/ml, respectively and their calculated SI values were 14.6, 18.4 and 14.1, respectively (Zandi et al., 2010).

Camellia sinensis
Tea is a cultivated evergreen plant and its green, oolong and black types are all made from the same plant species, Camellia sinensis L. Their differences in appearance, organoleptic taste, and chemical content as well as flavour are due to their fermentation process. The chemical components of its leaves include polyphenols (catechins and flavonoides), alkaloids (caffeine, theobromine, the ophylline, etc.), volatile oils, polysaccharides, amino acids, lipids, vitamins (e.g., vitamin C), inorganic elements (e.g., aluminium, fluorine and manganese), etc. The beneficial properties of tea are attributed to its polyphenols. Its flavonoides have antioxidant, antiinflammatory, anti-allergic and antimicrobial effects. The polyphenols content of green tea and black tea varies from 30% to 40% and 3% to 10%, respectively. Tea and particularly, green tea has been evaluated for its medicinal properties and a number of studies suggested that chemical constituents of tea play important role in human health (Sharangi, 2009). It was shown that in vitro replication of HSV-1 is inhibited by aqueous extract of C. sinensis with IC 50 value and selectivity index of 50 and 20 μg/mL, respectively (Farahani, 2013).

Euphorbia spinidens
Plants in the family of Euphorbiaceae are well known for the chemical diversity of their isoprenoid constituents (Jury et al., 1987). Members of the Euphorbia genus are known to contain substances with inhibitory action against some bacteria. This inhibitory action has been attributed to their large amounts of phenolic compounds (Li et al., 2009). E. spinidens is an indigenous plant naturally growing in different geographical regions of Iran and is increasingly used in traditional medicine. Crude methanol extract of E. spinidens and the corresponding fractions n-Hexane, Chloroform, and n-Butanol, with SI values of 15.85, 2.062, 8.2, and 25.37, respectively, inhibited early stages of HSV-1 replication, in vitro (Mohammadi-Kamalabadi et al., 2014).

Conclusion
Searching for new bioactive compounds in order to develop new anti-viral agents seems to be urgent. Research on some medicinal plants may potentially lead to isolation of active compounds which could be effective against HSV. To achieve this goal, both experimental and clinical investigations are needed to provide a better understanding of mechanisms of action, therapeutic effects, and the safety profile of these compounds. Iran with a diverse climate and wide variety of herbal flora could be considered as potentially a great source of these herbs from which isolation of these components is possible. Therefore, this systematic review which covered the published results of research on some Iranian medicinal plants and the corresponding isolated active phytochemicals, conclusively, would highlight the need for more comprehensive studies using more advanced methodology to develop promising anti-HSV agents from these bioactive compounds.